Serotonin has been linked to a broad array of behavioral and affective states, yet technological limitations have historically constrained the interrogation of this circuitry. Originating from the raphe nuclei, the serotonin system exhibits distinct projections to both cortical and subcortical networks, engaging in widespread communication with downstream targets. Selective serotonin reuptake inhibitors (SSRIs) are among the most commonly prescribed pharmacological treatments for depression/anxiety, yet it is mostly unknown how they impact endogenous serotonin function. Notably, SSRIs take approximately 2-4 weeks before they begin to exert antidepressant effects, indicating a neuromodulatory effect. Here, we present our lab’s improved iSeroSnFR2.0 fluorescent serotonin indicator, coupled with in vivo fiber photometry, to characterize the effects of the SSRI fluoxetine on endogenous serotonin activity. We examined the modulatory effects of acute (24 hours) and chronic (25 days) oral fluoxetine on serotonin dynamics in the orbitofrontal cortex (OFC) and bed nucleus of the stria terminalis (BNST) during Pavlovian aversive learning. We find that while acute fluoxetine increases serotonin response to a learned cue, chronic fluoxetine ablates cue-triggered release. We couple this with the use of machine learning to model the behavioral changes that occur across chronic fluoxetine administration. Our results serve a dual purpose of validating proprietary tools for in vivo use and demystifying the neuromodulatory impacts of SSRIs. We hope these findings contribute to a more thorough understanding of therapeutic interventions that systemically target serotonin circuitry, elucidating region-specific drug effects on neurochemical release.